Genetic engineering approaches have allowed the production of recombinant antibodies having specific binding specificities, specific domain structures, and other desirable properties. One type of genetically engineered antibody is the single chain Fv fragment (scFv). Single chain Fv fragments are genetically engineered polypeptides that contain a heavy chain variable region (VH) linked to a light chain variable region (VL) via a flexible peptide linker. Each VH and VL domain contains three complementarity determining regions (CDRs). CDRs are short amino acid sequences that vary greatly among antibody molecules, and thus, are responsible for generating the great diversity of antibody binding specificity. The combination of the CDRs of the VH plus the CDRs of the VL determines the binding specificity of any given antibody.
Single chain Fv fragments display the binding specificity and monovalent binding affinity of full-size antibodies and provide the added benefit of relative ease of genetic manipulation and expression (because scFvs are encoded by and expressed from a single coding sequence, rather than from separate coding sequences, as are full-size antibodies). Single chain Fv fragments and other recombinant antibodies are used in a broad variety of applications, for example, in medical diagnostic tests, in basic research, and as therapeutic antibody treatments for various diseases.
Intrabodies are genetically-engineered antibody molecules that are ectopically expressed within cells. Intrabodies can be used to visualize or to modulate the function of a target antigen within living cells. For example, the use of intrabodies can induce a phenotypic knockout either by directly inhibiting the function of the targeted antigen or by diverting the targeted antigen from its normal intracellular location (e.g., an intrabody can redirect its target antigen to the degradation machinery). Intrabodies can also enhance or change the function of their target antigens. For protein targets, intrabodies can be targeted to a specific post-translational modification or to a specific antigen conformation. Moreover, an intrabody-induced phenotypic knockout can be confined to a specific cell compartment by targeting an intrabody to the specific subcellular compartment using an addressing signal (e.g., a nuclear localization signal, a mitochondrial localization signal, or an endoplasmic reticulum retention signal). Intrabodies can also modulate target function by modifying the oligomeric structure of the target.
Because intrabody phenotypic knockout relies only on the binding capacity of the antibody molecule to its target, it is not necessary to express within the cell a complete antibody molecule but only its binding site, which is entirely located within the variable region (Fv). Given their advantages of small size and antigen specificity encompassed within a single polypeptide chain, scFvs are the most common type of recombinant antibody fragment used for intracellular antibody expression.
One serious limitation to the use of intrabodies is that most scFvs are not able to fold under the reducing conditions of the cell cytosol and nucleus. Under such conditions the two conserved disulfide bridges of scFvs are reduced, thereby destabilizing and inactivating the binding activity of many scFvs. In vitro, most scFvs cannot be renatured under reducing conditions. Statistical analyses of scFv sequences have shown that fewer than 1% of the scFvs are stable enough to be expressed and active in absence of disulfide bond formation. In addition, even if a scFv protein is indeed stable enough in its reduced form to be expressed and active in vivo, other parameters such as protease susceptibility or folding kinetics may also influence the final in vivo fate of the intrabody and thus are critical for ultimate intrabody expression and activity.
To obtain an active intrabody, current approaches often involve two successive steps. First, a panel of scFv or Fab antibodies that specifically bind an antigen of interest are identified (for example, by screening a phage display library). Second, the specifically-binding antibodies are tested for their ability to bind and/or inhibit the target antigen in vivo. Because fewer than 1% of scFvs are potentially useful as intrabodies (because they are not expressed and/or cannot properly fold under the reducing conditions that exist within a cell), identification of a single scFv that can be used as an intrabody requires the isolation of more than 100 scFv clones, a number that is unlikely to be obtained in most cases.
In view of the foregoing difficulties in producing and identifying antibodies that can be used as intrabodies for use in medical and research applications, what is needed are more efficient methods of producing and selecting antibodies that can be used as intrabodies.